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Endometriosis has been associated with a reduced response to progesterone in both the eutopic and ectopic endometrium. In this study we evaluated OVGP1 and steroid receptor expression in oviducts of baboons with endometriosis during the midsecretory phase and determined whether progesterone resistance associated with endometriosis also occurs in the oviduct. Oviducts obtained during the window of uterine receptivity (Day 10 postovulation [PO]) from animals with induced and spontaneous disease were compared to control animals during the proliferative stage and in the implantation window as well as animals treated with the progesterone receptor (PGR) antagonist ZK 137.299 (ZK). OVGP1 was significantly higher in animals with endometriosis compared with Day 10 PO controls and was similar to that seen in the late proliferative phase and in ZK-treated animals. Baboons with spontaneous endometriosis also showed a similar persistence of OVGP1, which was correlated with the maintenance of estrogen receptor 1 (ESR1) in the epithelial cells of animals with endometriosis. However, epithelial cell height and the percentage of ciliation were not affected by endometriosis. These data imply that the normal antagonism of progesterone on ESR and OVGP1, which results in their downregulation during the window of implantation, is absent in animals with endometriosis. This was confirmed further when the action of PGR was antagonized in animals without disease, which also resulted in the persistence of ESR1 and OVGP1. These studies suggest that an aberrant oviductal environment may be an additive factor that contributes to endometriosis-associated infertility.
Endometriosis is an enigmatic gynecological disease that is characterized by the presence of endometrial tissues outside of the uterine cavity. The incidence of endometriosis increases up to 30% in patients with infertility and up to 45% in patients with chronic pelvic pain [1, 2]. The spontaneous progressive nature of this disease has been demonstrated in 30%–60% of patients [3, 4]. Both circumstantial evidence and laboratory evidence indicate that estradiol and progesterone have critical roles in the establishment and maintenance of the disease. Endometriosis also has been associated with a blunted response to progesterone in eutopic uterus, and it has been suggested the resistance to progesterone action is related to the overall reduction of progesterone receptor (PGR) levels . Our studies in the baboon also suggest that functional PGR in the eutopic endometrium of baboons with endometriosis is altered during the window of uterine receptivity [6, 7]. Other studies have also suggested that decreases in the PGRB isoform in endometriotic tissues may also be an important mechanism that contributes to the progesterone resistance associated with the disease . Global gene expression analysis of the eutopic endometrium of women with endometriosis also suggests that the proliferative to secretory transition is dysregulated, resulting in an attenuated response due to reduced progesterone responsiveness as a consequence of the disease .
The ovarian steroid hormones estradiol and progesterone regulate the functions of the reproductive tract throughout the menstrual cycle. In humans and nonhuman primates, the cyclic changes in the expression of the estrogen receptor (ESR) and PGR in the endometrium during the various phases of the menstrual cycle have been well documented [10–12]. The oviduct is also a target tissue for the ovarian steroids. Cyclic changes of progesterone and estradiol control homeostasis of the oviduct epithelium as well as formation and beat frequency of the cilia . Like the endometrium, oviductal function is also governed by estradiol and progesterone. In the primate, estradiol promotes hypertrophy and ciliogenesis of the oviductal epithelium with increased levels of OVGP1 protein; in the secretory phase, increasing progesterone levels antagonizes the actions of estradiol. Furthermore, increased levels of oviductal ESR and PGR are present during the estrogenic proliferative phase, whereas progesterone decreases both ESR and PGR during the secretory phase .
Our laboratory has extensively studied the gene regulation and potential function of OVGP1 in both the baboon and human oviduct [14–16] and demonstrated that this high-molecular weight glycoprotein is exquisitely regulated by estradiol and antagonized by progesterone . Therefore, the objective of this study was to determine whether endometriosis antagonizes the actions of progesterone in the baboon oviduct as it does in the endometrium [6, 7, 17], which may further contribute to the infertility that is associated with endometriosis. Regulation of the secretion of OVGP1 was examined by both Western immunoblots and immunohistochemistry (IHC) and correlated with changes in ESR1 and PGR and with morphological features of the oviductal epithelial cells throughout the cycle.
The animals were normally cycling females ranging in age from 7 to 12 yr and weighing between 12 and 18 kg. The females were housed in individual cages in the Biological Research Laboratories of the University of Illinois. Control oviductal tissues were obtained from normally cycling baboons during the proliferative (n = 8) and secretory (n = 6) phases of the menstrual cycle . Endometriosis was experimentally induced in six female baboons with regular menstrual cycles by intraperitoneal inoculation of menstrual endometrium on two consecutive menstrual cycles, and oviductal samples were collected at necropsy between 12 and 17 mo after inoculation. Details of the inoculation procedure have been described previously [18–21]. In the colony two additional animals were diagnosed with spontaneous endometriosis. Their tissues were also collected as a comparative reference. As shown in Table 1, the induction of the disease did not significantly alter the length of the menstrual cycle. Tissues were also obtained from baboons treated with chorionic gonadotropin (CG) [19, 22] and from baboons (n = 3) treated with the PGR antagonist ZK137.299 (ZK) . In the CG-treated group, oviducts were obtained from four controls and from three baboons with induced endometriosis. The baboons treated with PGR antagonist were disease free. The rationale for analyzing these two treatment groups was based on our previous studies, which demonstrated that the effects of CG on the receptive endometrium are attenuated in animals with endometriosis. This response resembles that seen when PGR is antagonized in both cycling and CG-treated baboons during the window of uterine receptivity . Tissues from all groups were obtained during the window of uterine receptivity (Day 10 postovulation [PO]). Tissues from animals with induced endometriosis were obtained at the 15- to 16-mo time point, when the final surgery was done to obtain tissues based on the experimental paradigm used in our previous studies [21, 24]. All animal procedures were approved by the Animal Care Committee of the University of Illinois at Chicago.
Oviducts were dissected away from the uterus following the removal of the entire reproductive tract at necropsy and transported to the laboratory in cold Hanks buffered salt solution on ice. The harvested tissue was snap frozen in liquid nitrogen for protein extraction or fixed in either 10% buffered formalin or in Bouin fixative for 24 h at room temperature for IHC. The frozen tissues were processed for protein extraction using RIPA buffer (Pierce, Rockford, IL). The fixed tissue was embedded in paraffin, and 5-μm sections were used for IHC localization using specific antibodies against ESR1, PGR, and OVGP1 [6, 16]. Histological analyses for the epithelial cell height and the percentage of ciliated epithelial cells were also quantified on Gomori-stained slides . The Gomori stain is a one-step trichrome procedure that stains the cytoplasm red and collagen fibrils, including the connective tissues, green.
For IHC, the mouse full-length anti-human ESR1 antibody (NCL-L-ER-6F11; Vector Laboratories, Burlingame, CA) was diluted 1:40 in 1% horse serum. The rat anti-human PGR antibody (JZB39) was diluted at 1:50. The rabbit anti-baboon oviductal OVGP1 polyclonal antibody  was diluted at 1:8000. The PGR antibody was kindly provided by Dr. Geoffrey Greene of the University of Chicago (Chicago, IL).
For Western blot analysis, OVGP1 antibody was used at 1:20000 diluted in 1× PBS with 5% BSA and 0.1% Tween-20.
Formalin-fixed, paraffin-embedded ampulla sections (5 μm) were deparaffinized through a series of xylene and graded alcohol washes and rehydrated in distilled water. Sections for steroid receptors were subjected to antigen retrieval using the Decloaking Chamber electric pressure cooker (Biocare Medical, Walnut Creek, CA). Retrieval of antigens required submerging the slides in Antigen Unmasking Solution (Vector Laboratories) in the pressure cooker for 5 min under 17 to 25 psi at 120°C. Slides were cooled for 20 min; washed under running distilled, deionized water for 10 min; and then washed in Tris-buffered saline (TBS) for 10 min. Tissues for OVGP1 staining were not subjected to antigen retrieval. Oviduct sections from the proliferative stage of the menstrual cycle served as positive controls . Endogenous peroxidase activity was quenched in 0.3% hydrogen peroxide in methanol for 15 min at room temperature . Sections were blocked in normal serum for 30 min at room temperature.
Sections were incubated with primary antibodies overnight at 4°C. Incubation of sections with preimmune serum as negative controls was performed at the same concentrations as the immune serum. Slides were washed three times in TBS and incubated with biotinylated secondary antibodies for 30 min. After washing, the slides were incubated with avidin-biotin peroxidase complex (Vector Elite ABC kit; Vector Laboratories) for 30 min, and positively stained cells were visualized by 3,3-diaminobenzidine tetrahydrochloride dihydrate. Staining was analyzed with a Nikon E400 microscope, and the images were captured using a Spot 4.1 color cooled digital camera equipped with advanced image capture software (Diagnostic Instruments, Sterling Heights, MI) at magnifications of 40× or 100×.
The intensity of staining was assessed, and the proportion of positive cells was also assessed. The intensity was graded on a four-point scale: (0) negative, 1(+) weak, 2(++) moderate, and 3(+++) strong. The H score was calculated using the following equation: H-score=ΣPi (i + 1); P is the percentage of the positively stained cells, whereas i is the intensity of the stained cells .
A total of 300 cells from three different areas of each slide were assessed under the light microscope at magnification of 40× by two independent observers. The height of each consecutive epithelial cell with a nucleus was measured in micrometers across the middle of the nucleus. The cell height represents the mean value of the 300 epithelial cells on each slide. The number of ciliated cells that formed the proportion of 1000 epithelial cells was recorded. The percentage of ciliated cells that were present within this epithelial cell population was also calculated.
Total protein for Western blot analysis was obtained from oviductal tissues from control baboons (n = 6) and baboons with endometriosis (n = 3). Protein extracts (25 μg) were electrophoresed on a 10% polyacrylamide-SDS gel, and the separated proteins were transferred to polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA). Membranes were blocked overnight at 4°C in 1× PBS, 0.1% Tween 20, and 5% BSA with continuous agitation. Membranes were incubated with OVGP1 antibodies for 1 h at room temperature, washed in PBS with 0.1% Tween, and probed for 1 h at room temperature with a secondary peroxidase-conjugated goat anti-rabbit immunoglobulin G (Bio-Rad, Hercules, CA). Protein complexes were detected with a chemiluminescent detection kit (Amersham Biosciences, Piscataway, NJ), and membranes were exposed on Hyper film ECL (Amersham Life Sciences, Arlington Heights, IL).
The data from the H score analysis for ESR1, PGR, OVGP1, and oviduct epithelial cell height (micrometers) and the percentage of ciliated epithelial cells were analyzed using a Graph Pad Stat 3 software (Graph Pad Inc., San Diego, CA) and one-way ANOVA analysis with significance at P < 0.05.
OVGP1 changes were analyzed by both IHC (Figs. 1 and and2)2) and Western blot analysis (Fig. 3). In the disease-free control animals, the expression of OVGP1 was high (H score 2.04 ± 0.2) in the proliferative phase and decreased markedly in the midsecretory phase in both cycling, non-Cg-treated (0.78 ± 0.26), and CG-treated (0.58 ± 0.22) baboons (Fig. 1, A–C and Fig. 2, A–C). In contrast, the expression of OVGP1 in the midsecretory phase in baboons with endometriosis was as high as the proliferative phase controls in both groups of animals (2.13 ± 0.3 and 2.21 ± 0.51; Fig. 1, E and F and Fig. 2, E and F). This high-level expression of OVGP1 was also evident in the ZK-treated baboons during the midsecretory phase (1.89 ± 0.45; Figs. 1D and and22D).
The immunohistochemical studies were further confirmed by Western blot analysis. A strong immunoreactive band (130000 kDa)  was evident in protein extracts from the proliferative phase (Fig. 3, lane a) and was markedly decreased in control baboons during the midsecretory phase in the absence or presence of CG (Fig. 3, lanes b and f). In contrast, strong immunoreactive bands were evident in the oviducts from baboons with induced or spontaneous endometriosis (Fig. 3, lanes c and d) and endometriotic baboons treated with CG (Fig. 3, lane g). Antagonism of progesterone action with ZK137.299 also prevented the decrease in OVGP1 in cycling baboons during the midsecretory phase (Fig. 3, lane e).
The ESR1 and PGR were localized by IHC (Figs. 4 and and55 [ESR1] and Figs. 6 and and77 [PGR]). During the late proliferative stage, nuclear staining for ESR1 was intense in the epithelial cells (1.60 ± 0.07; Fig. 4A) and decreased during the secretory phase (Fig. 4, B and C) in normally cycling baboons. This was also associated with a change in the morphology of the epithelial cells following ovulation (Fig. 8). Treatment with CG slightly increased the intensity of ESR1 (2.05 ± 0.11; Fig. 4C), although ZK treatment had no effect (1.74 ± 0.09; Fig. 4D). In baboons with induced endometriosis, there was a marked increase in staining intensity for ESR in the absence (2.73 ± 0.02) or presence (2.60 ± 0.05) of CG, which was statistically quantifiable by H score analysis (P < 0.001; Fig. 5, E and F). This increase in ESR1 staining is associated with the increased staining intensity and IHC for OVGP1 in these tissues (Figs. 1 and and3).3). During the proliferative phase, PGR staining was most intense in the epithelial cells and, in contrast with ESR1, stromal cells also stained for PGR (Fig. 6A). Unlike ESR1 expression, however, PGR localization was uniformly decreased in the epithelial cells of both control and endometriotic animals at Day 10 PO, and stromal staining continued to be faint but evident in all groups analyzed (Fig. 6, B–F). The quantification of the PGR staining intensity in epithelial cells is shown in Figure 7. The changes in PGR correlated with the morphological changes (Fig. 8).
Histological analysis of epithelial cell height and the percentage of ciliated epithelial cells were quantified on Gomori-stained slides, and these results are summarized in Figure 8. During the proliferative phase, the epithelium consists of approximately equal numbers of columnar ciliated and secretory cells, and epithelial cell height is maximal (Fig. 8A). However, during the midsecretory phase, most of the epithelium consists primarily of cuboidal cells, and only 6.38 ± 2.39% are ciliated (Fig. 8B). In contrast to OVGP1 and ESR1, the presence of endometriosis or treatment with PGR antagonist did not prevent the decrease in ciliation or cell height during the midsecretory phase (Fig. 8, C–F).
Endometriosis is a common gynecological disorder that is associated with chronic pelvic pain and infertility. The etiology and pathogenesis of this disease and its association with reduced fecundity still remain unclear. Jackson et al.  reported recently that both ESR and PGRA expression patterns during the window of receptivity are altered in the baboons with experimentally induced endometriosis. ESR1 was maintained in the glandular epithelium of animals with endometriosis. Although no differences in PGR expression in the stromal cells of animals with disease were evident, their ability to respond to ligand stimulation was impaired, suggesting that during the window of receptivity, endometriosis results in the development of an endometrium that is resistant to progesterone stimulation . This resistance to progesterone during the proliferative to secretory transition has also been demonstrated in the endometrium of women with endometriosis, and global gene expression analysis has revealed an intrinsic biological attenuation suggestive of a reduced progesterone response in the transition from the proliferative to the secretory phase of the menstrual cycle .
At the time of ovulation, the primate oviduct consists of both columnar epithelial and secretory cells. Under estradiol dominance these cells are differentiated and functional while progesterone initiates their dedifferentiation. The secretory cells synthesize and secrete the glycoprotein OVGP1 in response to estradiol . Other studies have also suggested that alterations in PGR in endometriotic tissues may be an important mechanism that is responsible for progesterone resistance . Our data would suggest that a similar mechanism of progesterone resistance persists in the oviduct and is associated with the persistence of ESR1 in the epithelial cells and PGR in the stroma. However, unlike in the uterus, where the overexpression of estrogen-regulated genes such as FOS, CYR61, and BSG diminishes as the disease progresses , OVGP1 in the oviduct continues to persist in baboons with endometriosis.
OVGP1 is an estrogen-dependent protein synthesized by nonciliated oviduct epithelial cells that is secreted into the oviductal lumen in a variety of species, including baboon and human [14, 16, 26, 27]. During the normal menstrual cycle, OVGP1 is highly expressed in the secretory epithelial cells of the oviduct during the proliferative phase and is dramatically decreased following ovulation [26, 27]. This is correlated with the downregulation of ESR1. The synthesis of OVGP1 by the baboon oviduct is directly correlated with the presence or absence of the ovarian steroids, and it is estrogen dependent and oviduct specific . It is also specifically localized to the secretory granules of the nonciliated oviductal epithelial cells in humans and nonhuman primates [14–16]. Transient transfection studies of both the human and the mouse OVGP1 promoter suggest that the imperfect ERE found on both promoters is responsive to estradiol stimulation [28, 29]. To date, however, it has been reported that most estrogen-responsive genes contain one or more imperfect EREs or multiple copies of the ERE half-site rather than the consensus ERE. Previous studies have suggested that mammalian OVGP1 is secreted from oviductal epithelial cells in an estrogen-dependent manner , although the precise mechanism of its transcriptional regulation still remains controversial. OVGP1 has been shown to play an important role in enhancing sperm capacitation, sperm-zona binding, and penetration through the zona pellucida in several different species [13, 26]. More recent evidence also suggests that the expression of OVGP1 is increased in both ovarian and endometrial cancers, suggesting that OVGP1 could be a useful marker of early events associated with ovarian carcinogenesis [30, 31]. These studies also further suggest that reproductive tract pathologies are associated with an increase or persistence of OVGP1.
In this study OVGP1 expression was highest in the proliferative phase in control animals, as expected. It decreased markedly during the midsecretory phase in response to progesterone in both cycling and hCG-treated baboons. In contrast, OVGP1 expression was maintained during the midsecretory phase in animals with induced endometriosis; the staining intensity of OVGP1 in these animals was comparable to that present during the proliferative phase in control animals and correlated with the continued presence of ESR in the oviductal epithelial cells. We suggest that as a result of endometriosis, the oviduct, like the uterine endometrium develops progesterone resistance such that the ability of estradiol to continue to induce OVGP1 during the secretory phase is not impaired.
The evidence to support the development of progesterone resistance in woman and baboons with endometriosis is substantial [5, 6, 9, 17]. The eutopic endometrium has reduced responsiveness to progesterone, altered distribution of ESR and PGR isoforms, and the dysregulation of progesterone target genes [5, 6, 17, 32, 33]. Our data would suggest that this impaired responsiveness to progesterone is also manifested in the oviduct of baboons with endometriosis. This is further substantiated by the treatment of control baboons with the PGR antagonist ZK 137.299. Antagonism of PGR with a competitive inhibitor also prevented the downregulation of OVGP1 in the oviduct during the midsecretory phase. Furthermore, the eutopic endometrium of these animals shows a similar response to that observed in baboons with endometriosis treated with CG . Although the presence of endometriosis prevented the decrease in OVGP1 during the midsecretory phase, the morphological changes in the epithelial cells that are also steroid receptor mediated  were unaffected. These data imply that the stromal progesterone response, which modulates epithelial function , is not altered by the presence of ectopic lesions. OVGP1, however, is a direct estradiol-responsive gene [28, 29], and perhaps the continued presence of ESR1 in the epithelial cells antagonizes the ability of progesterone to downregulate gene expression.
In summary, these data further suggest that the inability of progesterone to regulate gene expression in the oviduct may alter the oviductal environment and contribute to endometriosis-associated infertility. These data also suggest that progesterone resistance associated with this disease in the eutopic endometrium is also further manifested in the oviduct results in physiological alterations in the entire reproductive tract that are inconsistent with the midsecretory phase of the menstrual cycle.
1Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development/ National Institutes of Health through cooperative agreement U54 HD 40093 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.